A polyglycol-modified lipid compound and its preparation method and application

Abstract

The present invention discloses a lipid compound modified with polyglycol, a preparation method and an application thereof. The lipid compound and lipid nanoparticles prepared therefrom can effectively deliver bioactive substances to target cells and parts in a targeted manner, and efficiently achieve the pharmacological effects of the bioactive substances. The lipid compound of the present invention has a single molecular weight, which is conducive to controlling batch differences, improving drug stability, and reducing immunogenicity, and is expected to be used in the development and application of related drugs.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a lipid compound modified by polyglycol, and a preparation method and application thereof. Background Art

[0002] Gene therapy refers to the use of molecular biological methods to introduce target genes into patients to make them express in order to correct or compensate for diseases caused by gene defects and abnormalities. It also refers to the introduction of nucleic acids into cells to inhibit the expression of target genes (gene silencing) or increase the expression of target genes (gene activation) to achieve the purpose of treating diseases. In recent years, with the advancement of biotechnology, gene therapy drugs have developed rapidly, bringing huge development prospects to the field of biological drugs. Alnylam, a US company, has developed a series of gene drugs using RNAi technology and successfully launched four RNAi drugs: Onpattro (patisiran), Givlaari (givosiran), Oxlumo (lumasiran) and Leavio (inclisiran). Among them, Leavio (inclisiran) is a biological drug for the treatment of hyperlipidemia. The successful launch of this drug has expanded the use of gene therapy drugs from the traditional field of treating rare genetic diseases to the field of treating mass diseases, bringing new opportunities for RNAi drugs.

[0003] Nucleic acid drugs cannot effectively penetrate the cell membrane and enter cells due to their own properties, such as negative charge and easy degradation by nucleases, and are quickly degraded in the body. Therefore, a suitable delivery system is needed to stably deliver nucleic acids to the target location and take effect. The common difficulty in the development of mRNA and siRNA is how to effectively deliver them to the cells at the target site. During the delivery process, it is also necessary to consider how to avoid rapid clearance, avoid degradation by nucleases, and improve escape after endocytosis.

[0004] At present, the systems for delivering nucleic acids mostly use different types of liposome delivery methods, such as lipid nanoparticles (LNP), GalNac, LPP (lipopolyplexes), etc. LNP can be prepared from four lipids in a certain ratio, usually including cationic lipids, neutral lipids, steroidal lipids and polymer-conjugated lipids, among which polymer-conjugated lipids refer to polyethylene glycol (PEG) lipids.

[0005] Some polyethylene glycol lipids are publicly reported in the prior art, for example, patent documents CN110352071A and CN1882693A disclose polyethylene glycol lipids and lipid nanoparticles prepared using polyethylene glycol lipids for delivering bioactive substances into the body. However, polyethylene glycol itself is a mixture as a polymer, which is prone to batch differences, and has an adverse effect on the quality and efficacy stability of the drugs formed by it. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the present invention provides a compound and a preparation method and application thereof.

[0007] In a first aspect of the present invention, a compound is provided, which has the following structure:

[0008]

[0009] in,

[0010] R 1 and R 2 Each is independently a hydrocarbon group containing 6 to 30 carbon atoms;

[0011] L 1 , L 2 and L 3 is an independent linking group;

[0012] X is N or CR 4 , where R 4 Selected from: H, alkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, halogen, -CN, -NO 2 、-COR A 、-C(O)OR A 、-OCOR A 、-C(O)NR A R B 、-CH=NR A 、-OR A 、-OC(O)R A 、-S(O) t -R A 、-S(O) t -NR A R B 、-NR A R B 、-NR A C(O)R B ; Wherein, t is an integer from 0 to 2 (such as 0, 1, 2), each R A and R B are independently selected from: H, alkyl, cycloalkyl, aryl, heterocyclyl and halogen;

[0013] n is an integer selected from 30-90;

[0014] Y is a terminal group.

[0015] The compound has a single molecular weight, rather than being a mixture of species of different molecular weights (such as polymers, which are generally characterized by an average molecular weight).

[0016] Specifically, L 1 and L 2 can be independently selected from: a single bond, an alkylene group, -OC(O)(CH 2 ) i -、-C(O)O(CH 2 ) i -、-C(O)(CH 2 ) i -、-O(CH 2 ) i -、-S(O) x (CH 2 ) i -、-SS-(CH 2 ) i -、-C(O)S(CH 2 ) i -、-SC(O)(CH 2 ) i -、-NR a -(CH 2 ) i -、-C(O)NR a -(CH 2 ) i -、-OC(O)NR a -(CH 2 ) i -、-NR a -C(O)(CH 2 ) i -、-NR a -C(O)O(CH 2 ) i -、-S(O) x NR a -(CH 2 ) i -、-OC(O)O(CH 2 ) i -; wherein x is an integer from 0 to 2 (such as 0, 1, 2), i is an integer from 0 to 10, and each R a are independently selected from the group consisting of: H, alkyl, cycloalkyl, aryl, heterocyclyl and halogen.

[0017] Specifically, i is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, especially 0 or 1.

[0018] Specifically, each R a are independently selected from: H, C1-6 alkyl; in some embodiments of the present invention, R a For H.

[0019] More specifically, L 1 and L 2 Can be independently selected from: single bond, C1-6 alkylene (e.g. -CH 2 -、-CH 2 CH 2 -、-CH 2 CH 2 CH 2 -), -OC(O)(CH 2 )-、-OC(O)-、-C(O)O(CH 2 )-、-C(O)O-、-C(O)(CH 2 )-、-C(O)-、-C(O)S(CH 2 )-、-C(O)S-、-C(O)NH-(CH 2 )-、-C(O)NH-、-OC(O)O(CH 2 )-、-OC(O)O-.

[0020] In some embodiments of the present invention, L 1 and L 2 All are single bonds.

[0021] In other embodiments of the present invention, L 1 -C(O)O(CH 2 )-,L 2 It is -C(O)O-.

[0022] Specifically, L 3 Can be selected from: single bond, alkylene, -(CH 2 ) h OC(O)(CH 2 ) j -、-(CH 2 ) h C(O)O(CH 2 ) j -、-(CH 2 ) h C(O)(CH 2 ) j -、-(CH 2 ) h O(CH 2 ) j-、-(CH 2 ) h S(O) y (CH 2 ) j -、-(CH 2 ) h SS-(CH 2 ) j -、-(CH 2 ) h C(O)S(CH 2 ) j -、-(CH 2 ) h SC(O)(CH 2 ) j -、-(CH 2 ) h NR b -(CH 2 ) j -、-(CH 2 ) h C(O)NR b -(CH 2 ) j -、-(CH 2 ) h OC(O)NR b -(CH 2 ) j -、-(CH 2 ) h NR b -C(O)(CH 2 ) j -、-(CH 2 ) h NR b -C(O)O(CH 2 ) j -、-(CH 2 ) h S(O) y NR b -(CH 2 ) j -、-(CH 2 ) h OC(O)O(CH 2 ) j -; wherein y is an integer of 0-2 (such as 0, 1, 2), h and j are independently selected from integers of 0-10, each R b are independently selected from the group consisting of: H, alkyl, cycloalkyl, aryl, heterocyclyl and halogen.

[0023] Specifically, h and j are independently selected from: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, especially 0 and 2; in some embodiments of the present invention, h is 0.

[0024] Specifically, each R b are independently selected from: H, C1-6 alkyl; in some embodiments of the present invention, R b For H.

[0025] More specifically, L 3 Can be selected from: single bond, C1-6 alkylene (e.g. -CH 2 -、-CH 2 CH 2 -、-CH 2 CH 2 CH 2 -), -OC(O)(CH 2 ) 2 -、-OC(O)-、-C(O)O(CH 2 ) 2 -、-C(O)O-、-C(O)(CH 2 ) 2 -、-C(O)-、-C(O)S(CH 2 ) 2 -、-C(O)S-、-C(O)NH-(CH 2 ) 2 -、-C(O)NH-、-OC(O)O(CH 2 ) 2 -、-OC(O)O-.

[0026] In some embodiments of the present invention, L 3 is C1-6 alkylene, such as -CH 2 -、-CH 2 CH 2 -、-CH 2 CH 2 CH 2 -.

[0027] In other embodiments of the present invention, L 3 -C(O)CH 2 CH 2 -.

[0028] In some embodiments of the present invention, L 1 , L 2 and L 3 It can be an uncleavable linker, which is relatively stable in the in vivo environment and is not easily cleaved, for example, a thioether linker, an alkylene linker, or a single bond directly connected to R1 , R 2 or Connect, especially C1-10 (straight or branched) alkylene, such as C2-8 alkylene, C2-5 alkylene, -CH 2 -、-CH 2 CH 2 -、-CH 2 CH 2 CH 2 -.

[0029] Specifically, R 1 and R 2 Each is independently a straight chain or branched, saturated or unsaturated hydrocarbon group containing 6 to 30 carbon atoms (e.g., 6, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30 carbon atoms), in particular a straight chain hydrocarbon group containing 6 to 30 carbon atoms; more specifically, R 1 and R 2 Each of them can independently be an alkyl group containing 6 to 30 carbon atoms, in particular a straight-chain alkyl group containing 6 to 30 carbon atoms, in particular a straight-chain alkyl group containing 10 to 24 carbon atoms, for example, C12 straight-chain alkyl group (lauryl), C14 straight-chain alkyl group (myristyl), C16 straight-chain alkyl group (palmityl), C18 straight-chain alkyl group (stearyl), C20 straight-chain alkyl group (eicosyl).

[0030] In some embodiments of the present invention, R 1 and R 2 Same, for example, R 1 and R 2 are straight chain alkyl groups containing 6 to 24 carbon atoms, for example, R 1 and R 2 All are C14 straight chain alkyl (myristyl).

[0031] Specifically, R 4 is selected from: H, C1-6 alkyl, halogen; in some embodiments of the present invention, R 4 For H.

[0032] Specifically, n can be 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 60, 65, 70, 75, 80, 85, 90; more specifically, n can be an integer of 30-60, for example, an integer of 40-50, an integer of 44-48; in some embodiments of the present invention, n is 46 or 47.

[0033] Specifically, Y can be selected from: H, alkyl, alkoxy, cycloalkyl, aralkyl, monosaccharide and oligosaccharide groups; More specifically, Y can be selected from: methyl, ethyl, isopropyl, methoxy, ethoxy, cyclopropyl, cyclobutyl, cyclohexyl, benzyl, In particular, Y is methyl, ethyl, isopropyl, methoxy, ethoxy, cyclopropyl, cyclobutyl, cyclohexyl or benzyl; in some embodiments of the invention, Y is methoxy.

[0034] Specifically, Y can also be selected from: hydroxyl, carboxyl, ester, amino, thiol, maleimide, succinimide, succinimide ester, alkynyl, azido, aldehyde, nitrobenzene carbonate, acrylate, methacrylate, dibenzocyclooctyne, isocyanate, vinyl sulfone, dithiopyridyl, glutaric acid, hydrazide, p-nitrocarbonate, silane, epoxy, etc.

[0035] Specifically, the compound may have the following structure:

[0036]

[0037] Among them, L 1 , L 2 , L 3 , R 1 , R 2 , n, and Y have the corresponding definitions as defined above in the present invention.

[0038] More specifically, the compound can be selected from the following structures:

[0039]

[0040] Among them, R 1 , R 2 , n, and Y have the corresponding definitions as defined above in the present invention.

[0041] In some embodiments of the present invention, the compound has the following structure:

[0042]

[0043] In the second aspect of the present invention, provided are pharmaceutically acceptable salts, esters, isomers, prodrugs and solvates of the compound described in the first aspect.

[0044] In the third aspect of the present invention, a method for preparing the compound of the first aspect is provided, which may include using The step of carrying out the reaction, wherein n and Y have the corresponding definitions in the first aspect of the present invention, R c is a reactive group, for example, a hydroxyl group, a carboxyl group, an amino group, a thiol group, a maleimide group, a succinimide group, an alkynyl group, an azide group, an aldehyde group, an epoxy group, and the like.

[0045] In a fourth aspect of the present invention, a lipid composition is provided, comprising the compound described in the first aspect or a pharmaceutically acceptable salt, ester, isomer, prodrug and solvate thereof.

[0046] Specifically, the lipid composition may also include cationic lipids such as octadecanoylamide (SA), lauryl trimethylammonium bromide, hexadecyl trimethylammonium bromide, myristyl trimethylammonium bromide, dimethyl di-octadecanoyl bromide (DDAB), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), 1,2-di-tetradecanoyl-3-trimethylammonium-propane (DMTAP), 1,2-di-octadecanoyl-3-trimethylammonium-propane (DOTAP) and DOTAP derivatives such as 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane and 1,2-di-hexadecenoyl-3-trimethylammonium-propane, 1,2-di-(9Z-octadecenoyl)-3-dimethylammonium-propane (DODAP) and DODAP derivatives such as Such as 1,2-di-tetradecanoyl-3-dimethylammonium-propane, 1,2-di-hexadecanoyl-3-dimethylammonium-propane and 1,2-di-octadecanoyl-3-dimethylammonium-propane, 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-c-(4'-trimethylammonium)-butyryl-sn-glycerol (DOTB), di-octadecanoyl-alanyl spermine, SAINT-2, polycationic lipid 2,3-dioleoyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propylammonium trifluoroacetate (DOSPA), ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), etc. One or more, especially ALC-0315.

[0047] Specifically, in the lipid composition, the molar ratio of the compound described in the first aspect or its pharmaceutically acceptable salt, ester, isomer, prodrug and solvate to the cationic lipid can be 1:0.01-0.1 (for example, 1:0.01, 1:0.02, 1:0.025, 1:0.03, 1:0.035, 1:0.04, 1:0.045, 1:0.05, 1:0.06, 1:0.08, 1:0.1), in particular 1:0.03-0.05.

[0048] Specifically, the lipid composition can also contain neutral lipids, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), etc. One or more, especially DSPC.

[0049] Specifically, in the lipid composition, the molar ratio of the compound described in the first aspect or its pharmaceutically acceptable salt, ester, isomer, prodrug and solvate to the neutral lipid can be 1:0.1-0.5 (for example, 1:0.1, 1:0.15, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4, 1:0.5), in particular 1:0.1-0.3.

[0050] Specifically, the lipid composition can also contain steroidal lipids, such as avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, black sea sterol, epicholesterol, ergosterol, fucosterol, hexahydroluminosterol, hydroxycholesterol; lanosterol, luminosterol, alginosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid, etc. One or more, especially cholesterol.

[0051] Specifically, in the lipid composition, the molar ratio of the compound described in the first aspect or its pharmaceutically acceptable salt, ester, isomer, prodrug and solvate to the steroidal lipid can be 1:0.5-1.5 (1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.4, 1:1.5), especially 1:0.8-1.2.

[0052] More specifically, the lipid composition comprises the compound described in the first aspect, as well as cationic lipids, neutral lipids and steroidal lipids; in some embodiments of the present invention, the composition comprises the compound described in the first aspect, as well as ALC-0315, DSPC and cholesterol.

[0053] Specifically, the lipid composition may further comprise one or more pharmaceutically acceptable excipients, such as carriers, adjuvants, diluents, and the like.

[0054] In the fifth aspect of the present invention, a pharmaceutical composition is also provided, which comprises a biologically active substance and the compound described in the first aspect or the lipid composition described in the third aspect.

[0055] Specifically, the bioactive substance can be a small molecule compound, nucleic acid, peptide, protein, etc.

[0056] Specifically, the bioactive substance is a nucleic acid, such as DNA or RNA, wherein the DNA can be non-coding DNA (antisense DNA) or coding DNA, and the RNA can be selected from: one or more of antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, gRNA, tsRNA, etc., especially mRNA and siRNA.

[0057] In one embodiment of the present invention, the bioactive substance is siRNA that inhibits the expression of the target gene, such as anti-KDR siRNA.

[0058] Specifically, the bioactive substance can be used to prevent and / or treat one or more diseases selected from the following: cancer, inflammation, fibrotic diseases, autoimmune diseases, diseases caused by pathogen infection, mental disorders, blood diseases, chromosomal diseases, genetic diseases, connective tissue diseases, digestive diseases, ear, nose and throat diseases, endocrine diseases, eye diseases, reproductive diseases, heart diseases, kidney diseases, lung diseases, metabolic disorders, oral diseases, musculoskeletal diseases, nutritional diseases, skin diseases and the like.

[0059] Specifically, in the pharmaceutical composition, the molar ratio of the biologically active substance to the compound described in the first aspect or the lipid composition described in the third aspect is 0.01-0.5:1 (for example, 0.01:1, 0.02:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1), especially 0.01-0.1.

[0060] Specifically, the pharmaceutical composition may further include one or more pharmaceutically acceptable excipients, such as carriers, adjuvants, diluents, and the like.

[0061] In some embodiments of the present invention, the pharmaceutically acceptable excipients are pharmaceutically acceptable injection excipients, such as isotonic sterile salt (sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc., or a mixture of the above salts) solution.

[0062] Specifically, the pharmaceutical composition can be administered by any suitable route, such as gastrointestinal administration or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intraorgan, intranasal, intradermal, instillation, intracerebral, rectal, etc.); the drug can be in any suitable dosage form, such as a dosage form for gastrointestinal administration, for example, including, but not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, etc.; a dosage form for parenteral administration, for example, injection Dosage forms: such as injections (for example, for subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), respiratory tract administration forms: such as sprays, aerosols, powder sprays, etc., skin administration forms, such as external solutions, lotions, ointments, plasters, pastes, patches, etc., mucosal administration forms: such as eye drops, eye ointments, nasal drops, gargles, sublingual tablets, etc., cavity administration forms: such as suppositories, aerosols, effervescent tablets, drops, pills, etc., for rectum, vagina, urethra, nasal cavity, ear canal, etc. In particular, injection dosage forms.

[0063] Specifically, various dosage forms of the above-mentioned pharmaceutical composition can be prepared according to conventional production methods in the pharmaceutical field.

[0064] In the sixth aspect of the present invention, there is provided the compound described in the first aspect or its pharmaceutically acceptable salts, esters, isomers, prodrugs and solvates, and the lipid composition described in the fourth aspect, for use in the preparation of a delivery system for biologically active substances and in the delivery of biologically active substances.

[0065] Specifically, in this application, the biologically active substance has the definition as described in the fourth aspect of the present invention.

[0066] Specifically, the delivery system of the above-mentioned bioactive substances is lipid nanoparticles (LNP).

[0067] In the seventh aspect of the present invention, provided is the use of the compound described in the first aspect or its pharmaceutically acceptable salts, esters, isomers, prodrugs and solvates, the lipid composition described in the fourth aspect, and the pharmaceutical composition described in the fifth aspect in the preparation of drugs for preventing and / or treating diseases, and in preventing and / or treating diseases.

[0068] Specifically, the disease can be selected from one or more of the following: cancer, inflammation, fibrotic disease, autoimmune disease, disease caused by pathogen infection, mental illness, blood disease, chromosomal disease, genetic disease, connective tissue disease, digestive disease, ear, nose and throat disease, endocrine disease, eye disease, reproductive disease, heart disease, kidney disease, lung disease, metabolic disease, oral disease, musculoskeletal disease, nutritional disease, skin disease, etc.

[0069] Specifically, the pathogen may be a microorganism, a parasite (protozoa, worms, etc.) or other agents; wherein the microorganism may be selected from one or more of: viruses, chlamydia, rickettsia, mycoplasma, bacteria, spirochetes, fungi, etc.

[0070] Specifically, viruses include, but are not limited to, adenoviridae (such as adenovirus), herpesviridae (such as HSV1 (oral herpes), HSV2 (genital herpes), VZV (varicella), EBV (Epstein-Barr virus), CMV (cytomegalovirus)), poxviridae (such as smallpox virus, vaccinia virus), papovaviridae (such as papillomavirus (HPV)), parvoviridae (such as B19 virus), hepadnaviridae (such as hepatitis B virus), polyomaviridae (such as polyomavirus), reoviridae (such as reovirus, rotavirus), picornaviridae (such as enterovirus, foot-and-mouth disease virus), caliciviridae (such as Norwalk virus, hepatitis E virus), togaviridae (such as rubella virus), arenaviridae (such as lymphocytic choriomeningitis virus), retroviridae (HIV-1, HIV-2, HTLV-1), Flaviviridae (such as dengue virus, Zika virus , Japanese encephalitis virus, Chikungunya virus, yellow fever virus, hepatitis C virus, West Nile virus, etc.), Orthomyxoviridae (such as influenza virus (such as influenza A virus, influenza B virus, influenza C virus, etc.)), Paramyxoviridae (such as human parainfluenza virus type 1 (HPIV), HPIV type 2, HPIV type 3, HPIV type 4, Sendai virus, mumps virus, measles virus, respiratory syncytial virus, Newcastle disease virus, etc.), Bunyaviridae (such as California encephalitis virus, Hantavirus), Rhabdoviridae (such as rabies virus), Filoviridae (such as Ebola virus, Marburg virus), Coronaviridae (such as HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, etc.), Astroviridae (such as astrovirus), Bornaviridae (such as Borna virus) viruses.

[0071] In some embodiments of the present invention, the virus is a coronavirus, such as HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, SARS-CoV-2, etc.

[0072] In some embodiments of the present invention, the virus is an orthomyxovirus, such as an influenza virus (eg, influenza A virus, influenza B virus, influenza C virus, etc.).

[0073] In some embodiments of the present invention, the virus is a paramyxovirus, such as HPIV type 1, HPIV type 2, HPIV type 3, HPIV type 4, Sendai virus, mumps virus, measles virus, respiratory syncytial virus, etc.

[0074] In some embodiments of the present invention, the virus is a flavivirus, such as dengue virus, Zika virus, Japanese encephalitis virus, chikungunya virus, yellow fever virus, hepatitis C virus, West Nile virus, etc.

[0075] In some embodiments of the invention, the virus is papillomavirus (HPV).

[0076] Specifically, diseases caused by viral infection include, but are not limited to, influenza, SARS, COVID-19, viral hepatitis (such as hepatitis A, hepatitis B, hepatitis C, hepatitis D, etc.), mumps, measles, dengue fever, Zika virus disease, Japanese encephalitis, chikungunya disease, yellow fever, West Nile virus disease, cervical cancer, etc.

[0077] In some embodiments of the present invention, the above-mentioned drug is a drug for preventing a disease, such as a vaccine.

[0078] In other embodiments of the present invention, the above-mentioned drug is a therapeutic agent for treating a disease.

[0079] In the eighth aspect of the present invention, a method for delivering a biologically active substance is provided, which comprises the steps of preparing a delivery system for the biologically active substance with the compound described in the first aspect or its pharmaceutically acceptable salts, esters, isomers, prodrugs and solvates or the lipid composition described in the fourth aspect, and then delivering the biologically active substance to a subject in need thereof through the delivery system.

[0080] Specifically, in this method, the biologically active substance has the definition as described in the fourth aspect of the present invention.

[0081] Specifically, the biologically active substance is encapsulated in the delivery system.

[0082] Specifically, in this method, the subject can be any animal or cell thereof (in vitro or in situ) that is subjected to the method; more specifically, the subject is a mammal, for example, a rat, a mouse, a guinea pig, a rabbit, a dog, a monkey or a human, in particular a human.

[0083] In the ninth aspect of the present invention, a method for preventing and / or treating a disease is provided, which comprises the steps of administering the pharmaceutical composition of the fifth aspect to a subject in need thereof, or preparing a delivery system for a bioactive substance using the compound of the first aspect or its pharmaceutically acceptable salts, esters, isomers, prodrugs and solvates or the lipid composition of the fourth aspect, and then delivering the bioactive substance to a subject in need thereof through the delivery system, or adopting the method for delivering a bioactive substance of the eighth aspect.

[0084] In particular, the subject is a mammal, for example, a rat, a mouse, a guinea pig, a rabbit, a dog, a monkey or a human, in particular a human.

[0085] After the lipid nanoparticles (LNPs) prepared by the lipid compound of the present invention deliver anti-KDR siRNA (siRNA that inhibits the expression of VEGFR2 mRNA) into cells, they have a significant and sustained inhibitory effect on KDR mRNA, indicating that the lipid compound and the lipid nanoparticles prepared therefrom can effectively deliver the bioactive substance to the target cells and parts in a targeted manner, and efficiently achieve the pharmacological effects of the bioactive substance. In addition, the lipid compound of the present invention has a single molecular weight, which is conducive to controlling batch differences, improving drug stability, and reducing immunogenicity, and is expected to be used in the development and application of related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 Shown is the mass spectrum of compound 1 prepared in Example 1 of the present invention.

[0087] Figure 2 Shown is the mass spectrum of compound 2 prepared in Example 2 of the present invention.

[0088] Figure 3 Shown is the mass spectrum of compound 3 prepared in Example 3 of the present invention. DETAILED DESCRIPTION

[0089] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0090] In the present invention, the terms "VEGFR2" and "KDR" both represent Vascular Endothelial Growth Factor Receptor 2 and can be used interchangeably.

[0091] In the present invention, the term "alkyl" refers to a straight or branched hydrocarbon group that does not contain unsaturated bonds, and the hydrocarbon group is connected to the other parts of the molecule with a single bond. The alkyl used in this article usually contains 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms (i.e., C1-10 alkyl), preferably contains 1 to 6 carbon atoms (i.e., C1-6 alkyl). The example of alkyl includes but is not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, n-hexyl, isohexyl, etc. If the alkyl is replaced by a cycloalkyl, it is correspondingly a "cycloalkylalkyl", such as cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, etc. If the alkyl is replaced by an aryl, it is correspondingly a "aralkyl", such as benzyl, benzhydryl or phenethyl. If the alkyl is replaced by a heterocyclic radical, it is correspondingly a "heterocyclic radicalalkyl".

[0092] In the present invention, the term "alkylene" refers to a hydrocarbon group (divalent alkyl) formed by losing two hydrogen atoms from an alkane molecule, which can be a straight chain or a branched chain and is connected to the rest of the molecule by a single bond. The alkyl group used in this article usually contains 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms (i.e., C1-10 alkylene), and particularly contains 1 to 6 carbon atoms (i.e., C1-6 alkylene). Examples of alkylene include methylene (-CH 2 -), ethylene (-CH 2 CH 2 -), propylene, butylene, etc.

[0093] In the present invention, the term "alkenyl" refers to a straight or branched hydrocarbon group containing at least two carbon atoms and at least one unsaturated bond, and the hydrocarbon group is connected to the other parts of the molecule by a single bond. The alkenyl used in this article usually contains 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms (i.e., C1-10 alkenyl), preferably contains 1 to 6 carbon atoms (i.e., C1-6 alkenyl). Examples of alkenyl include, but are not limited to, vinyl, 1-methyl-vinyl, 1-propenyl, 2-propenyl or butenyl, etc.

[0094] In the present invention, the term "cycloalkyl" refers to alicyclic hydrocarbons. The cycloalkyl used in this article usually contains 1 to 4 single rings and / or condensed rings, 3-18 carbon atoms, preferably 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms (e.g., C3-10 cycloalkyl, C3-6 cycloalkyl), such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl or adamantyl, etc.

[0095] In the present invention, the term "aryl" refers to any functional group or substituent derived from a simple aromatic ring, including monocyclic aryl groups and / or condensed aryl groups, such as 1-3 rings, monocyclic or condensed rings, and having 6-18 (e.g., 6, 8, 10, 12, 14, 16, 18) carbon ring atoms. The aryl used herein is generally an aryl (i.e., C6-12 aryl) containing 1-2 rings, monocyclic or condensed rings and having 6-12 carbon ring atoms, wherein the H on the carbon atom can be substituted, for example, by groups such as alkyl, halogen, etc. Examples of the aryl include, but are not limited to, phenyl, p-methylphenyl, naphthyl, biphenyl, indenyl, etc.

[0096] In the present invention, the term "halogen" refers to bromine, chlorine, iodine or fluorine.

[0097] In the present invention, the term "heterocyclyl" refers to a 3- to 18-membered non-aromatic ring group containing 2 to 17 carbon atoms and 1 to 10 heteroatoms. The heterocyclyl can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can contain fused, spirocyclic or bridged ring systems. The heterocyclyl can be partially saturated (heteroaryl) or fully saturated (heterocycloalkyl). Suitable heteroaryl groups in the compounds of the present invention contain 1, 2 or 3 heteroatoms selected from N, O or S atoms, and include, for example, coumarin, including 8-coumarin, quinolyl, including 8-quinolyl, isoquinolyl, pyridinyl, pyrazinyl, pyrazolyl, pyrimidinyl, furanyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, triazolyl, tetrazolyl, isoxazolyl, oxazolyl, imidazolyl, indolyl, isoindolyl, indazolyl, indolizinyl, phthalazinyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, pyridazinyl, triazinyl, cinnolinyl, benzimidazolyl, benzofuranyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl and furopyridinyl. Suitable heterocycloalkyl groups in the compounds of the present invention contain 1, 2 or 3 heteroatoms selected from N, O or S atoms, and include, for example, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, piperidinyl, morpholinyl, thiomorpholinyl, oxathiolanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxirane, thiirane, azepine, oxazepine, diazepine, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, dihydroindolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothiolanyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexyl, 3-azabicyclo[4.1.0]heptyl, 3H-indolyl and quinolizinyl.

[0098] The pharmaceutically acceptable salts of the present invention include acid addition salts and base addition salts.

[0099] The acid addition salts include, but are not limited to, salts from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid and phosphonic acid, and salts from organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids and aliphatic and aromatic sulfonic acids. Therefore, these salts include but are not limited to sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, iodate, acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate and mesylate, also comprising amino acid salts such as arginate, gluconate, galacturonate etc. Acid addition salts can be prepared by contacting the free alkali form with a sufficient amount of the required acid to form a salt in a conventional manner. The free alkali form can be regenerated by contacting the salt form with an alkali, and the free alkali is separated in a conventional manner.

[0100] Base addition salts of the present invention refer to salts formed with metals or amines, such as hydroxides of alkali metals and alkaline earth metals, or with organic amines. Examples of metals used as cations include, but are not limited to, sodium, potassium, magnesium, and calcium. Examples of suitable amines include, but are not limited to, N, N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (ethane-1,2-diamine), N-methylglucamine, and procaine. Base addition salts can be prepared by contacting the free acid form with a sufficient amount of the desired base in a conventional manner to form a salt. The free acid form can be regenerated by contacting the salt form with an acid, and the free acid is separated in a conventional manner.

[0101] In the present invention, stereoisomers include enantiomers, diastereomers and geometric isomers. Some compounds of the present invention have cycloalkyl groups, which can be substituted on more than one carbon atom, in which case all geometric forms, including cis and trans, and mixtures thereof, are within the scope of the present invention. The cycloalkyl groups include alicyclic hydrocarbon groups and aryl groups, wherein the alicyclic hydrocarbon groups can be non-aromatic monocyclic, condensed ring, bridged ring or spirocyclic saturated or unsaturated cyclic hydrocarbon groups, and aryl groups such as phenyl, naphthyl, phenanthrenyl, biphenyl, etc.

[0102] In the present invention, solvate refers to the physical combination of the compound of the present invention and one or more solvent molecules. The physical combination includes various degrees of ionic and covalent bonding, including hydrogen bonding. In some cases, the solvate can be separated, for example, when one or more solvent molecules are incorporated into the lattice of the crystalline solid. Solvates include solution phases and separable solvates. Representative solvates include ethanolates, methanolates, etc.

[0103] In the present invention, prodrugs refer to forms of the compounds of formula I which are suitable for administration to patients without excessive toxicity, irritation, allergic reactions, etc. and are effective for their intended use, including acetal, ester and zwitterion forms. Prodrugs are transformed in vivo, such as by hydrolysis in the blood, to obtain the parent compound.

[0104] In the present invention, the term "nucleic acid" refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in a single-stranded or double-stranded form, and includes DNA, RNA, and hybrids thereof.

[0105] In the present invention, the term "lipid" refers to a group of organic compounds, which include but are not limited to esters of fatty acids, and are generally characterized by being poorly soluble in water but soluble in many organic solvents.

[0106] In the present invention, the term "cationic lipid" refers to a lipid molecule that is capable of carrying a positive charge.

[0107] In the present invention, the term "neutral lipid" refers to uncharged, non-phosphoglyceride lipid molecules.

[0108] In the present invention, the term "lipid nanoparticle" refers to a particle having at least one dimension of the nanometer order, which comprises at least one lipid.

[0109] In the present invention, the term "vaccine" refers to a composition suitable for application to animals (particularly mammals, such as humans), which induces an immune response after administration, the strength of which is sufficient to minimally help prevent, improve or cure clinical diseases caused by microbial infection.

[0110] In the present invention, the term "delivery system" refers to a preparation or composition that regulates the distribution of biologically active ingredients in space, time and dosage in the body.

[0111] In the present invention, the term "patient" or "subject" and the like are used interchangeably herein and refer to any animal or cell thereof treated according to the methods described herein, which may be in vitro or in situ. Specifically, the aforementioned animal includes mammals, for example, rats, mice, guinea pigs, rabbits, dogs, monkeys or humans, in particular humans.

[0112] In the present invention, the term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, suppressing and / or stopping one or more clinical symptoms of a disease after the onset of the disease.

[0113] In the present invention, the term "prevention" refers to avoiding, minimizing or making it difficult for a disease to occur or develop by treatment before the disease occurs.

[0114]

[00136] Various publications, patents, and published patent specifications are cited herein, the disclosures of which are incorporated by reference in their entireties.

[0115] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0116] Example 1: Synthesis of Compound 1

[0117]

[0118] 1. Synthesis of Compound 1a

[0119]

[0120] Tetradecylamine (3.0 g, 14 mmol) and tetradecyl bromide (3.9 g, 14 mmol) were dissolved in 20 ml of acetonitrile, potassium carbonate (1.9 g, 14 mmol) was added, and the mixture was stirred at 80°C for 12 hours. After the reaction of the raw materials was complete, the mixture was cooled to room temperature, filtered, and the residue was washed with dichloromethane. A saturated sodium bicarbonate solution was added to the obtained filtrate, and the mixture was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, and separated by column chromatography to obtain compound 1a (3.9 g, white solid) with a yield of 68%. MS m / z (ESI): 410.40 [M+1].

[0121] 2. Synthesis of Compound 1

[0122]

[0123] Compound 1a (0.576 g, 1.4 mmol) and (2g, 1mmol) was added to a 100ml single-mouth bottle containing dichloromethane (20ml), stirred to dissolve, and then DIEA (260mg, 2mmol) and HATU (760mg, 2mmol) were added in sequence, and stirred at room temperature for 5 hours. The reaction system was filtered, the mother liquor was concentrated in vacuo, 40ml of purified water was added after concentration, and it was stirred and dissolved and washed with ethyl acetate 3 times, using 40ml each time (the washing process will emulsify, and a small amount of ethanol is added for demulsification). After washing, 6g of sodium chloride was added to the aqueous phase and stirred to dissolve, and the aqueous phase was extracted with dichloromethane twice, using 20ml each time. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated in vacuo, 30ml of isopropanol and 10ml of ethyl acetate were added, and dissolved under heating conditions of 40°C, cooled to minus 15°C for crystallization, and filtered. The filter cake was vacuum dried to obtain the product compound 1 (1.5g, white solid) with a yield of 62.5%. MS m / z(ESI):2582.76[M+NH 4 ] + ;

[0124] 1H-NMR (300MHz, DMSO) δ: 2.96 (2H, s), 3.1~3.8 (180H, m), 1.46 (4H, m), 1.24 (44H, m), 0.86 (6H, t).

[0125] Example 2: Synthesis of Compound 2

[0126]

[0127] Under nitrogen atmosphere, LiAlH 4 (380mg, 10mmol) was slowly added to a three-necked flask containing 30ml of tetrahydrofuran in an ice-water bath. Compound 1 (2g, 1mmol) was dissolved in 10ml of tetrahydrofuran and slowly added dropwise to the reaction flask. After 3 hours, 10ml of purified water was added dropwise to the reaction system. The filtrate was concentrated in vacuo using a diatomaceous earth filtration system. 20ml of isopropanol and 6ml of ethyl acetate were added and dissolved under heating conditions of 40°C. The temperature was cooled in an ice-water bath to crystallize and filtered. The filter cake was vacuum dried to obtain the product compound 2 (1.3g, white solid) with a yield of 64%. MS m / z(ESI):2551.76[M+H] + ;

[0128] 1H-NMR (300MHz, DMSO) δ: 3.2~3.8(180H,m), 2.36(4H,t), 2.26(4H,m), 1.53(2H,m), 1.22(44H,m), 0.84(6H,t).

[0129] Example 3: Synthesis of Compound 3

[0130]

[0131] 1. Synthesis of compound 3a

[0132]

[0133] Will (40g, 20mmol) and purified water (0.4ml) were added to a 1L three-necked flask containing THF (400ml), stirred to dissolve, and then NaOH (6.4g, 160mmol) and epichlorohydrin (37g, 400mmol) were added in sequence, and heated to reflux for 18 hours. NaOH (3.2g, 80mmol) and epichlorohydrin (18.5g, 200mmol) were added and the reaction was continued for 18 hours. The reaction system was cooled to room temperature, filtered with diatomaceous earth, and pH = 7 phosphate buffer (100ml) was added to the mother liquor. THF was removed by vacuum concentration, and purified water (200ml) was added after concentration. Wash with ethyl acetate twice, using 100ml each time. After washing, 45g of sodium chloride was added to the aqueous phase and stirred to dissolve, and the aqueous phase was extracted twice with dichloromethane, using 100ml each time. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated in vacuo, and 400 ml of isopropanol was added for recrystallization. The filter cake was filtered and dried in vacuo to obtain the product compound 3a (34 g, light yellow solid), with a yield of 85%. MS m / z (ESI): 2114.41 [M+H] + ;

[0134] 1H-NMR (300MHz, DMSO) δ: 3.3~3.85(184H,m), 3.28(1H,t), 3.28(3H,s), 3.1(1H,m), 2.7(1H,t), 2,55(1H,m).

[0135] 2. Synthesis of compound 3b

[0136]

[0137] Compound 3a (30 g, 15 mmol) was dissolved in 2 mol / L potassium hydroxide solution (300 ml) and stirred at room temperature for 12 hours. 45 g of sodium chloride was added and stirred to dissolve. The aqueous phase was extracted twice with dichloromethane, using 150 ml each time. The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was vacuum concentrated, 450 ml of methyl tert-butyl ether was added to precipitate a solid, filtered, and the filter cake was vacuum dried to obtain the product compound 3b (28 g, light yellow solid), with a yield of 93.3%. MS m / z (ESI): 2132.43 [M+H] + ;

[0138] 1H-NMR (300MHz, DMSO) δ: 4.6 (1H, s), 4.45 (1H, s), 3.3~3.8 (189H, m), 3.25 (3H, s).

[0139] 3. Synthesis of Compound 3

[0140]

[0141] Compound 3b (20g, 10mmol) was added to a 500ml single-mouth bottle containing DCM (200ml), stirred to dissolve, and tetradecanoic acid (6.4g, 28mmol) and DMAP (489mg, 4mmol) were added in turn and placed in an ice-water bath. DCC (5.8g, 28mmol) was dissolved in 40ml DCM and added dropwise to the reaction system. After 16 hours, the reaction system was filtered, the mother liquor was vacuum concentrated to remove dichloromethane, 300ml of isopropanol and 100ml of ethyl acetate were added, heated to 40°C to dissolve, cooled to minus 10°C for crystallization, and filtered. The filter cake was vacuum dried to obtain the product compound 3 (17g, light yellow solid) with a yield of 85%. MS m / z (ESI): 2569.70 [M+NH 4 ] + ;

[0142] 1H-NMR(300MHz,DMSO)δ:5.1(1H,s),4.28(1H,m),4.1(1H,m),3.3~3.8(1 83H,m),3.24(3H,s),2.25(4H,t),1.5(4H,m),1.24(44H,m),0.86(6H,t).

[0143] Example 4: Gene knockdown effect of siRNA:polyethylene glycol lipid (PEG-lipid) particle composition after cell delivery in ARPE-19 cells

[0144] 1. Cell culture and transfection

[0145] Cell name: ARPE-19

[0146] (1) ARPE-19 cells were cultured in DMEM / F12+12% FBS+dual antibody culture medium (containing 100 U / ml Penicillin, 100 μg / ml Streptomycin) at 37°C and 5% CO 2 The cells were cultured in a saturated humidity incubator. 24 h before the experiment, 1.2 × 10 5 Cells were seeded in 12-well plates and cultured overnight.

[0147] (2) Take 50 μL OPTI-MEM medium and dilute 2.5 mL anti-KDR siRNA: compound 1 = 1:10, anti-KDR siRNA: compound 2 = 1:10, anti-KDR siRNA: compound 3 = 1:10 (the above ratios are molar ratios, and the siRNA concentration is 20 μM). For the positive control, take 50 μL OPTI-MEM medium and dilute 3 μL Lipofectamine 3000 TM The transfection reagent was added and the two were mixed, gently shaken, and allowed to stand for 15 min. In addition, blank group, NC group, and NC-RL group were set up.

[0148] Compound 1, Compound 2 and Compound 3 were prepared from Examples 1-3, respectively.

[0149] Anti-KDR siRNA refers to siRNA that inhibits the expression of VEGFR2 mRNA. Its preparation method is described in patent document CN202010229195.2, and the sequence is:

[0150] Sense strand: 5′-GGAGUGAGAUGAAGAAAUU-3′;

[0151] Antisense strand: 5′-AAUUUCUUCAUCUCACUCC-3′.

[0152] (3) The cells in each well of the cell plate were replaced with medium without antibiotics, with a volume of 900 μL per well, and then 100 μL of the above-mentioned mixture was added to each well, with a final total volume of 1000 μL per well. The transfection concentration of siRNA (or siRNA NC or siR-NC RL) was 50 nM.

[0153] (4) 24h and 48h after transfection, place the 12-well plate in a 37°C, 5% CO 2 The cells were taken out of the incubator and collected for RNA extraction for subsequent detection.

[0154] 2. RNA Extraction

[0155] (1) RNA was extracted using the RNA extraction kit of Promega. In brief, after washing the cells with PBS, 300 μL of lysis buffer was added, and the cells were blown with a pipette. After sufficient lysis, 300 μL of diluent was added, and then the cells were placed in a water bath at 70°C for 3 min. Centrifuge at 14000 rpm for 10 min, transfer the supernatant to a new 1.5 mL Eppendorf tube, add 300 μL of anhydrous ethanol, mix thoroughly, and add to the adsorption column. Centrifuge at 14000 rpm for 1 min, discard the filtrate, add 600 μL of washing buffer, and centrifuge again for 1 min. Discard the filtrate, add 50 μL of freshly prepared DNase reaction solution to each well, and let stand at room temperature for 15 min; add 600 μL of washing buffer and centrifuge for 1 min; discard the filtrate and add 600 μL of washing buffer and centrifuge again for 1 min; discard the filtrate, centrifuge for 2 min, add 50 μL of nuclease-free water to each well, let stand at room temperature for 5 min, and then centrifuge to collect the eluted RNA.

[0156] (2) RNA quality inspection: Nanodrop was used to detect RNA content and purity, and 1% agarose gel electrophoresis was used to detect RNA integrity.

[0157] 3. Q-PCR detection process

[0158] (1) RNA reverse transcription

[0159] Using the total RNA extracted from the sample as a template and the Promega reverse transcription kit, the following reaction system was established:

[0160] Table 1 RNA reverse transcription system

[0161]

[0162] Mix the above system, collect the liquid to the bottom of the tube by centrifugation, and incubate at 42°C for 60 min and 72°C for 10 min; the product is the cDNA template.

[0163] (2) Fluorescence quantitative PCR detection

[0164] Using TB green Premix Ex Taq II (Tli RNaseH Plus) (Promega) reagent, the reaction system was established as follows:

[0165] Table 2 Fluorescence quantitative PCR reaction system

[0166]

[0167] PCR amplification was performed as follows

[0168] Pre-denaturation at 95℃ for 10min, then enter the following cycle

[0169] *95℃10s

[0170] 60℃20s

[0171] 70℃10s

[0172] Plate reading

[0173] Return* for a total of 40 cycles.

[0174] Prepare melting curve: between 65℃ and 95℃, read the plate every 0.5℃ and pause for 5s.

[0175] 4. Gene inhibition effect

[0176] GAPDH was used as the internal reference gene, and the relative expression of KDR mRNA was calculated by the ΔΔCt method. The expression level of the blank group was set as 100%, and the mRNA expression levels of each group were standardized.

[0177] The results showed that the relative expression level of KDR mRNA in the cells treated with siRNA of the present invention was significantly decreased compared with the blank group, siNC group and siNC RL group at 24 hours, and the expression was further decreased at 48 hours after transfection, indicating that the particles composed of compounds 1-3 of the present invention and siRNA have a significant and sustained inhibitory effect on KDR mRNA.

[0178] Example 5: Gene inhibition effect of siRNA:PEG-lipid particle composition after cell delivery in A375 cells

[0179] The above operations were similar to those in Example 4, except that the culture medium of A375 cells was DMEM culture medium containing 10% FBS (containing 100 U / ml Penicillin, 100 μg / ml Streptomycin). The results showed that the relative expression level of KDR mRNA in the cells treated with siRNA of the present invention was significantly decreased compared with the blank group, siNC group and siNCRL group at 24 hours, and further decreased at 48 hours after transfection, indicating that the particles (Compound 1, Compound 2, Compound 3) prepared by PEG-lipid and siRNA of the present invention had a significant and sustained inhibitory effect on KDR mRNA.

[0180] Example 6: Gene inhibition effect of PEG-lipid / cationic lipid / neutral lipid / steroid lipid-siRNA nanoparticles (LNP) after cell delivery in ARPE-19 cells

[0181] The formula and preparation method of each group of PEG-lipid / cationic lipid / neutral lipid / steroid lipid-siRNA nanoparticles (LNP) are as follows:

[0182] Compound 1 / ALC-0315 / DSPC / cholesterol was dissolved in anhydrous ethanol at a molar ratio of 46.3% / 1.7% / 9.4% / 42.6% to prepare a 10 mmol / L mixed solution, and then a pH = 4 citric acid buffer was added to prepare a 30% ethanol-citric acid solution containing four lipids, which was filtered with a 0.1 μm filter membrane for later use. Anti-KDR siRNA was dissolved in a lipid-free 30% ethanol-citric acid solution at a concentration of 2 mg / ml, and then mixed with the above 30% ethanol-citric acid solution containing four lipids at a mass ratio of anti-KDR siRNA: lipid of 0.06:1, incubated for 30 minutes, dialyzed with PBS at pH = 7.4 for more than 16 hours, and freeze-dried to obtain lipid nanoparticles containing anti-KDR siRNA. The preparation method of other groups of different PEG lipid ion lipid nanoparticles (Compound 1 replaced by Compound 2 or Compound 3) was the same as Compound 1.

[0183] The results show that after the anti-KDR siRNA is delivered into cells using each group of lipid nanoparticles prepared by the PEG-lipid / ALC-0315 / DSPC / cholesterol and siRNA of the present invention, the KDR mRNA has a significant and sustained inhibitory effect.

[0184] Example 7: Gene expression effect after cell delivery of PEG-lipid / cationic lipid / neutral lipid / steroid lipid-mRNA nanoparticles in 293T cells

[0185] The formula and preparation method of each group of PEG-lipid / cationic lipid / neutral lipid / steroid lipid-siRNA nanoparticles (LNP) are as follows:

[0186] Compound 1 / ALC-0315 / DSPC / cholesterol was dissolved in anhydrous ethanol at a molar ratio of 46.3% / 1.7% / 9.4% / 42.6% to prepare a 10 mmol / L mixed solution, and then a citric acid buffer solution with a pH of 4 was added to prepare a 30% ethanol-citric acid solution containing four lipids, which was filtered with a 0.1 μm filter membrane for later use. GFP mRNA was dissolved in a lipid-free 30% ethanol-citric acid solution at a concentration of 2 mg / ml, and then mixed with the above 30% ethanol-citric acid solution containing four lipids at a mass ratio of GFP mRNA: lipid of 0.06:1, incubated for 30 minutes, dialyzed with PBS with a pH of 7.4 for more than 16 hours, and freeze-dried to obtain lipid nanoparticles containing GFP mRNA. The preparation methods of other groups of different cationic lipid nanoparticles (Compound 1 replaced by Compound 2 and Compound 3) were the same as those of Compound 1.

[0187] The transfection effect of LNP was determined by detecting the number of cells expressing green fluorescent protein (GFP) using a fluorescence microscope. The results showed that compounds 1-3 as PEG lipid LNP groups all had good delivery effects.

[0188] Example 8: Study on the allergic reaction of PEG-lipids with different molecular weights in guinea pigs

[0189] 1. Animal grouping

[0190] 48 healthy guinea pigs that were normally raised for 5 days were randomly divided into 6 groups, 8 in each group. Group A: negative control group (0.9% sodium chloride injection); Group B: positive control group (0.15 mg / mL ovalbumin solution, ovalbumin purchased from Sigma, USA, batch number DHO15-4); Group C: M-PEG47-DTDPAM (i.e., compound 1, prepared in Example 1), Group D: M-PEG47-DTDPA (i.e., compound 2, prepared in Example 2), Group E: M-DTDPAM-2000 (provided by Tianjin Jiankai Technology Co., Ltd., batch number ZZ409P002), Group F: M-DTDPA-2000 (provided by Tianjin Jiankai Technology Co., Ltd., batch number ZZ409P004).

[0191] 2. Method of administration

[0192] Sensitization was performed by intraperitoneal injection, with a dosing volume of 1 mL / pig, once every other day, for a total of 4 times. On the second day after the last sensitization, each group of guinea pigs was stimulated by injection of the corresponding drug solution through the toe vein, with a stimulation dose of 2 mL / pig. The animal status was observed daily during the sensitization period, and the guinea pigs' reactions were observed continuously for 15 minutes before intravenous injection and for 40 minutes after injection, and the time of symptom onset and disappearance was recorded. Blood was collected 40 minutes after the stimulation was stopped, and heparin anticoagulation was used to prepare plasma.

[0193] 3. Observation indicators

[0194] During the sensitization period and the provocative drug administration, the reaction symptoms and duration of the animals were observed and recorded, such as scratching the nose, sneezing, restlessness, jumping, panting, purpura, etc. The score and level of the reaction symptoms were determined according to the systemic sensitization reaction symptom grading standard (Table 3).

[0195] Table 3 Grading criteria for systemic allergic reaction symptoms

[0196]

[0197] 4. Experimental results:

[0198] (1) Systemic allergy test results

[0199] Guinea pig reaction symptoms: No abnormal symptoms were observed in the animals of each group during the first sensitization. During the sensitization period, there were no abnormalities in diet, drinking water, and behavior, and the weight increased normally. No animals in group A showed allergic reactions after the provocative administration; strong allergic reactions occurred in group B after the provocative administration, manifested as spasms, purpura, unstable gait, panting, jumping, tears, and mental depression; 3 guinea pigs in group C showed occasional scratching and restlessness 10 to 35 minutes after the provocative administration, and 2 animals in group D showed the above symptoms. 4 guinea pigs in group E showed symptoms such as sneezing, coughing, and rapid breathing, and 1 showed symptoms such as erect hair and trembling; 3 guinea pigs in group F showed symptoms such as sneezing, coughing, and rapid breathing, and 2 guinea pigs showed symptoms of trembling and scratching the nose; the other guinea pigs were normal. According to Table 3, the reaction symptom level of guinea pigs in each group was judged as follows: group B was extremely positive, groups C and D were weakly positive, and groups E and F were positive. However, the frequency and degree of allergic reactions in groups C and D were lower than those in groups E and F. This suggests that the sensitization of single molecular weight PEG-lipid to guinea pigs is lower than that of non-single molecular weight PEG-lipid. The results of the grading of the reaction symptoms of guinea pigs in each group after provocative administration are shown in Table 4.

[0200] Table 4 Results of allergic reactions in guinea pigs caused by PEG-lipids with different molecular weights

[0201] Code Group Average rating Allergy symptoms Group A Negative control group 0 Negative Group B Positive control group 2.8 Strong positive Group C M-PEG47-DTDPAM 0.375 Weak positive Group D M-PEG47-DTDPA 0.25 Weak positive Group E M-DTDPAM-2000 1.125 Positive Group F M-DTDPA-2000 1 Positive

[0202] (2) Comparison of plasma IgE and histamine levels

[0203] After the stimulation administration, the plasma IgE levels of animals in each group were higher than those in group A, and the degree of increase was less than 100%. The plasma IgE levels of animals in groups C and D were significantly lower than those in groups E and F.

[0204] The plasma histamine levels in each medication group were higher than that in group A, and the histamine content in group B was significantly different from that in group A (P<0.05); the plasma histamine content in groups E and F was significantly higher than that in group A, with statistical differences. Although the plasma histamine content in groups C and D increased, it was much lower than that in groups E and F, and there was no statistical difference compared with group A.

[0205] Table 5 IgE and histamine levels in plasma of each group of guinea pigs after provocative administration (ng / ml)

[0206] Code Group IgE content (ng / ml) Histamine content (ng / ml) Group A Negative control group 204.83±18.65 4.92±0.98 Group B Positive control group 286.33±34.67 12.21±4.25 Group C M-PEG47-DTDPAM 235±26.79 5.23±1.21 Group D M-PEG47-DTDPA 225±31.44 6.71±2.10 Group E M-DTDPAM-2000 255±25.78 8.89±2.54 Group F M-DTDPA-2000 248±34.11 8.23±2.23

[0207] Conclusion: The results of the guinea pig systemic allergy test showed that non-single molecular weight PEG-lipids were positive, while single molecular weight PEG-lipids were weakly positive, indicating that compared with non-single molecular weight PEG-lipids, single molecular weight PEG-lipids have lower immunogenicity. The results of plasma IgE and histamine content tests also support this conclusion.

[0208] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0209] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience and preference of those skilled in the art.

[0210] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

Claims

1. A compound of single molecular weight having the following structure: in, R1 and R2 are independently selected from: C12 straight chain alkyl, C13 straight chain alkyl, C14 straight chain alkyl, C16 straight chain alkyl, C18 straight chain alkyl, C20 straight chain alkyl; n is 46, 47 or 48; Y is methoxy.

2. The compound according to claim 1, characterized in that R1 and R2 are both C14 straight chain alkyl, or, R1 and R2 are both C13 straight chain alkyl.

3. The compound according to claim 1, characterized in that The compound has the following structure:

4. A lipid composition comprising the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

5. The composition according to claim 4, characterized in that The composition also comprises a cationic lipid.

6. The composition according to claim 5, characterized in that The cationic lipid is selected from the group consisting of: octadecanoylamide (SA), lauryl trimethylammonium bromide, hexadecyl trimethylammonium bromide, myristyl trimethylammonium bromide, dimethyl dioctadecanoyl bromide (DDAB), 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-cholesterol), 1,2-di-tetradecanoyl-3-trimethylammonium-propane (DMTAP), 1,2-di-octadecanoyl-3-trimethylammonium-propane (DOTAP) and DOTAP derivatives, 1,2-di-(9Z-octadecenoyl)-3-dimethylammonium-propane (DODAP) and DO One or more of DAP derivatives, 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2-dioleoyl-c-(4'-trimethylammonium)-butyryl-sn-glycerol (DOTB), di-octadecyl-alanyl spermine, SAINT-2, polycationic lipid 2,3-dioleoyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanium trifluoroacetate (DOSPA), ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315); The DOTAP derivative is selected from: 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane and 1,2-di-hexadecanoyl-3-trimethylammonium-propane; The DODAP derivative is selected from the group consisting of: 1,2-di-tetradecanoyl-3-dimethylammonium-propane, 1,2-di-hexadecanoyl-3-dimethylammonium-propane and 1,2-di-octadecanoyl-3-dimethylammonium-propane.

7. The composition according to claim 5, characterized in that The cationic lipid is ALC-0315.

8. The composition according to claim 5, characterized in that The molar ratio of the compound to the cationic lipid is 1:0.01-0.

1.

9. The composition according to claim 4, characterized in that The composition also includes a neutral lipid.

10. The composition according to claim 9, characterized in that The neutral lipid is selected from: 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), oleoylphosphatidylcholine (POPC), and 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE).

11. The composition according to claim 9, characterized in that The neutral lipid is DSPC.

12. The composition according to claim 9, characterized in that The molar ratio of the compound to the neutral lipid is 1:0.1-0.

5.

13. The composition according to claim 4, characterized in that The composition also comprises a steroidal lipid.

14. The composition according to claim 13, characterized in that The steroidal lipids are selected from: avenasterol, β-sitosterol, brassicasterol, ergocalciferol, campesterol, cholestanol, cholesterol, coprosterol, dehydrocholesterol, streptosterol, dihydroergocalciferol, dihydrocholesterol, dihydroergosterol, melanosterol, epicholesterol, ergosterol, fucosterol, hexahydroluminosterol, hydroxycholesterol; lanosterol, luminosterol, alginosterol, sitostanol, sitosterol, stigmasterol, stigmasterol, bile acid, glycocholic acid, taurocholic acid, deoxycholic acid and lithocholic acid. One or more of.

15. The composition according to claim 13, characterized in that The steroidal lipid is cholesterol.

16. The composition according to claim 13, characterized in that The molar ratio of the compound to the steroidal lipid is 1:0.5-1.

5.

17. The composition of claim 4, comprising the compound, and ALC-0315, DSPC, and cholesterol.

18. A pharmaceutical composition comprising a biologically active substance and the compound according to any one of claims 1 to 3 or the lipid composition according to any one of claims 4 to 17, wherein the biologically active substance is DNA or RNA.

19. The composition according to claim 18, characterized in that The RNA is selected from: one or more of antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, gRNA, and tsRNA.

20. The composition according to any one of claims 18 to 19, characterized in that The molar ratio of the bioactive substance to the compound or the lipid composition is 0.01-0.5:

1.

21. The composition according to any one of claims 18 to 19, characterized in that The molar ratio of the bioactive substance to the compound or the lipid composition is 0.01-0.1:

1.

22. Use of the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, or the lipid composition according to any one of claims 4 to 17 in the preparation of a delivery system for a biologically active substance, wherein the biologically active substance is DNA or RNA.

23. The use according to claim 22, characterized in that The RNA is selected from: one or more of antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, gRNA, and tsRNA.

24. The use according to any one of claims 22 to 23, characterized in that: The delivery system of the bioactive substance is lipid nanoparticles (LNP).

25. Use of the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, the lipid composition according to any one of claims 4 to 17, or the pharmaceutical composition according to any one of claims 18 to 21 in the preparation of a medicament for preventing and / or treating a disease.

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